Many methods for peptide synthesis are described in the literature (for example, see U.S. Pat. No. 6,015,881; Mergler et al. (1988) Tetrahedron Letters 29:4005-4008; Mergler et al. (1988) Tetrahedron Letters 29:4009-4012; Kamber et al. (eds), Peptides, Chemistry and Biology, ESCOM, Leiden (1992) 525-526; Riniker et al. (1993) Tetrahedron Letters 49:9307-9320; Lloyd-Williams et al. (1993) Tetrahedron Letters 49:11065-11133; and Andersson et al. (2000) Biopolymers 55:227-250. The various methods of synthesis are distinguished by the physical state of the phase in which the synthesis takes place, namely liquid phase or solid phase.
In solid phase peptide synthesis (SPPS), an amino acid or peptide group is bound to a solid support resin. Then, successive amino acids or peptide groups are attached to the support-bound peptide until the peptide material of interest is formed. The support-bound peptide is then typically cleaved from the support and subject to further processing and/or purification. In some cases, solid phase synthesis yields a mature peptide product; in other cases the peptide cleaved from the support (i.e., a “peptide intermediate fragment”) is used in the preparation of a larger, mature peptide product.
Peptide intermediate fragments generated from solid phase processes can be coupled together in the solid phase or in a liquid phase synthetic process (herein referred to as “solution phase synthesis”). Solution phase synthesis can be particularly useful in cases where the synthesis of a useful mature peptide by solid phase is either impossible or not practical. For example, in solid phase synthesis, longer peptides eventually may adopt an irregular conformation while still attached to the solid support, making it difficult to add additional amino acids or peptide material to the growing chain. As the peptide chain becomes longer on the support resin, the efficiency of process steps such as coupling and deprotection may be compromised. This, in turn, can result in longer processing times to compensate for these problems, in addition to incremental losses in starting materials, such as activatable amino acids, co-reagents, and solvents. These problems can increase as the length of the peptide increases.
Therefore, it is relatively uncommon to find mature peptides of greater than 30 amino acids in length synthesized in a single fragment using only a solid phase procedure. Instead, individual fragments may be separately synthesized on the solid phase, and then coupled in the solid and/or solution phase to build the desired peptide product. This approach requires careful selection of fragment candidates. While some general principles can guide fragment selection, quite often empirical testing of fragment candidates is required. Fragment strategies that work in one context may not work in others. Even when reasonable fragment candidates are uncovered, process innovations may still be needed for a synthesis strategy to work under commercially reasonable conditions. Therefore, peptide synthesis using hybrid schemes are often challenging, and in many cases it is difficult to predict what problems are inherent in a synthesis scheme until the actual synthesis is performed.
In solution phase coupling, two peptide intermediate fragments, or a peptide intermediate fragment and a reactive amino acid, are coupled in an appropriate solvent, usually in the presence of additional reagents that promote the efficiency and quality of the coupling reaction. The peptide intermediate fragments are reactively arranged so the N-terminal of one fragment becomes coupled to the C-terminal of the other fragment, or vice versa. In addition, side chain protecting groups, which are present during solid phase synthesis, are commonly retained on the fragments during solution phase coupling to ensure the specific reactivity of the terminal ends of the fragments. These side chain protecting groups are typically not removed until a mature peptide has been formed.
Modest improvements in one or more steps in the overall synthetic scheme can amount to significant improvements in the preparation of the mature peptide. Such improvements can lead to a large overall saving in time and reagents, and can also significantly improve the purity and yield of the final product.
While the discussion of the importance of improvements in hybrid synthesis is applicable to any sort of peptide produced using these procedures, it is of particular import in the context of peptides that are therapeutically useful and that are manufactured on a scale for commercial medical use. Synthesis of larger biomolecular pharmaceuticals, such as therapeutic peptides, can be very expensive. Because of the cost of reagents, synthesis time, many synthesis steps, in addition to other factors, very small improvements in the synthetic process of these larger biomolecular pharmaceuticals can have a significant impact on whether it is even economically feasible to produce such a pharmaceutical. Such improvements are necessary due to these high production costs for larger biomolecular pharmaceuticals as supported by the fact that, in many cases, there are few, if any, suitable therapeutic alternatives for these types of larger biomolecular pharmaceuticals.
This is clearly seen in the case of the glucagon-like peptide-1 (GLP-1) and its counterparts. These peptides have been implicated as possible therapeutic agents for the treatment of type 2 non-insulin-dependent diabetes mellitus as well as related metabolic disorders, such as obesity. Gutniak, M. K., et al., Diabetes Care 1994:17:1039-44.
Lopez et al. determined that native GLP-1 was 37 amino acid residues long. Lopez, L. C., et al., Proc. Natl. Acad. Sci. USA., 80:5485-5489 (1983). This determination was confirmed by the work of Uttenthal, L. O., et al., J. Clin. Endocrinal. Metabol., 61:472-479 (1985). Native GLP-1 may be represented by the notation GLP-1 (1-37). This notation indicates that the peptide has all amino acids from 1 (N-terminus) through 37 (C-terminus). Native GLP-1 (1-37) has the amino acid sequence according to SEQ ID NO. 1:
HDEFERHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG
It has been reported that native GLP-1 (1-37) is generally unable to mediate insulin biosynthesis, but biologically important fragments of this peptide do have insulinotropic properties. For example, the native 31-amino acid long peptide GLP-1 (7-37) according to SEQ ID NO. 2
HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG
is insulinotropic and has the amino acids from the 7 (N-terminus) to the 37 (C-terminus) position of native GLP-1. GLP-1 (7-37) has a terminal glycine. When this glycine is absent, the resultant peptide is still insulinotropically active and is referred to as GLP-1 (7-36) according to SEQ ID NO. 3:
HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR
GLP-1 (7-36) often exists with the C-terminal arginine in amidated form, and this form may be represented by the notation GLP-1 (7-36)-NH2.
GLP-1 (1-37) generally is converted into an insulinotropically active counterpart thereof in vivo. For instance, GLP-1 (1-37) is naturally converted to GLP-1 (7-37) in vivo. This peptide, in turn, can also undergo additional processing by proteolytic removal of the C-terminal glycine to produce GLP-1 (7-36), which often exists in the amidated form GLP-1(7-36)-NH2. Accordingly, therapeutic treatments may involve administration of GLP-1 (1-37) or a counterpart thereof, with the expectation that an insulinotropically active derivative thereof forms in vivo. More commonly, however, therapeutic treatments under investigation involve administration of the insulinotropically active GLP-1 fragments themselves.
According to U.S. Pat. No. 6,887,849, the insulinotropic activity of GLP-1(7-37), GLP-1(7-36) and GLP-1(7-36)-NH2 appears to be specific for the pancreatic beta cells, where these peptides appear to induce biosynthesis of insulin. This makes these peptides and pharmaceutically acceptable counterparts thereof useful in the study of the pathogenesis of adult onset diabetes mellitus, a condition characterized by hyperglycemia in which the dynamics of insulin secretion are abnormal. Moreover, these glucagon-like peptides would be useful in the therapy and treatment of this disease, and in the therapy and treatment of hyperglycemia. According to EP 1137667 B1, these peptides or pharmaceutically acceptable counterparts thereof may also be useful for treating other types of diabetes, obesity, glucagonomas, secretory disorders of the airway, metabolic disorder, arthritis, osteoporosis, central nervous system disease, restenosis, neurodegenerative disease, renal failure, congestive heart failure, neophrotic syndrome, cirrhosis, pulmonary edema, hypertension, and/or disorders where a reduction in food intake is desired.
Native GLP-1 (1-37) and the native, insulinotropically active counterparts thereof according to SEQ ID NO. 1 through 3 are metabolically unstable, having a plasma half-life of only 1 to 2 minutes in vivo. Exogenously administered GLP-1 also is rapidly degraded. This metabolic instability has limited the therapeutic potential of native GLP-1 and native fragments thereof.
Synthetic counterparts of the GLP-1 peptides with improved stability have been developed. For instance, the peptide according to SEQ ID NO. 4 is described in EP 1137667 B1:
HAibEGTFTSDVSSYLEGQAAKEFIAWLVKAibR
This peptide is similar to the native GLP-1 (7-36), except that the achiral residue of alpha-aminoisobutyric acid (shown schematically by the abbreviation Aib) appears at the 8 and 35 positions in place of the corresponding native amino acids at these positions. The achiral alpha-aminoisobutric acid also is known as methylalanine. This peptide may be designated by the formula (Aib8,35)GLP-1 (7-36) or, in amidated form, (Aib8,35)GLP-1 (7-36)-NH2.
EP 1137667 B1 states that the peptide according to SEQ ID NO. 4 and its counterparts can be built as a single fragment using solid phase techniques. The single fragment synthesis approach suggested by EP 1137667 B1 is problematic. As one issue, this approach may lead to high levels of epimerization in the final amino acid coupling, e.g., histidine in the case of (Aib8,35)GLP-1 (7-36) for instance. Additionally, impurities may be hard to remove during chromatographic purification, and the yield may tend to be too low. Consequently, improved strategies for synthesizing peptides according to SEQ ID NO. 4 are needed in order to be able to manufacture this peptide and counterparts thereof in commercially acceptable yields, purities, and quantities.
In addition to these concerns, issues relating to product recovery and product purity for the large-scale production of peptides, as well as reagent handling, storage and disposal, can greatly impact the feasibility of the peptide synthesis scheme. Thus, there is a continuing need for peptide synthesis processes capable of efficiently producing peptide materials of commercial interest in large batch quantities with improved yields.